MiRNAs have emerged as a powerful class of conserved noncoding RNAs that regulate gene expression post- transcriptionally and play critical roles in numerous aspects of neuronal development and synaptic function. Impaired expression and/or function of miRNAs is implicated in several neurological diseases. A major gap is our poor understanding on the localization of specific microRNAs to the axon growth cone, and whether they play important roles to regulate local protein synthesis dependent effects on growth cone motility and guidance. The objectives of this application are to explore the new concept that microRNAs (miRNAs) localized to growth cones provide a regulated molecular mechanism to influence local protein synthesis underlying cue mediated and local protein synthesis dependent axon guidance. We will test the hypothesis that growth cone microRNAs regulate local translation to modulate attractive versus repulsive steering. Quantitative fluorescent in situ hybridization on cultured cortical neurons will be used to assess possible growth cone localization for microRNAs enriched in axonal fractions. We propose approaches to visualize the localization of microRNAs in cultured cortical neurons, to manipulate axonal microRNAs and target mRNAs translation in microfluidic devices, and image fluorescent reporters for local translation and growth cone guidance in live neurons. Aim 1 will identify miRNAs that are localized to axons by profiling microRNAs from axonal fractions of cortical neuron balls and visualization of their localization using fluorescent in situ hybridizatin. Aim 2 will examine the effects of manipulating axonal microRNA levels and function on axon outgrowth, growth cone morphology and steering responses to attractive and repulsive cues. Aim 3 will examine the effects of select candidate microRNAs on axonal protein expression and local protein synthesis in growth cones using immunofluorescence and live cell imaging of fluorescent reporters. The proposed research will advance our limited understanding of growth cone localized miRNAs and elucidate their mechanistic role in local protein synthesis underlying growth cone guidance. This research is expected to motivate studies to investigate the function of axonal microRNAs in the development of the nervous system in vivo, as well as dysfunction of micoRNA regulation leading to neurological diseases. These studies have important implications for future therapeutic strategies to modulate microRNA expression or function to manipulate axonal growth and connectivity in the treatment of neurologic disorders.